Enzymes have excellent catalytic functions with substrate specificity, reaction specificity, and stereospecificity. Stereospecificity of enzymes, with some exceptions, are nearly absolute.
Recent precise research has increased the importance of optically active substances for use in drugs, pesticides, feeds, and perfumes. Optical isomers sometimes have quite different biological activities; for example, D(R)-form thalidomide has no teratogenic activity, while its L(S)-form shows strong teratogenicity. Thus, the practical use of the thalidomide racemate caused the drug injury incidents by thalidomide. Furthermore, if one enantiomer shows an effective biological activity, the other enantiomer sometimes not only has no activity but moreover competitively inhibits the activity of the effective enantiomer. As a result, the biological activity of the racemate is reduced to half or less of the activity of the effective enantiomer. Accordingly, it is industrially important to obtain (synthesize or optically resolve) optically pure enantiomers. For this objective, an effective procedure has been used widely to optically resolve racemates synthesized. In particular, enzymatic optical resolution has drawn attention because it does not produce by-products and a bulk of liquid waste.
Generally, L-amino acids are widely and largely utilized in seasonings, food and feed additives, and infusions, and are thus very highly demanded. L-amino acids have been produced mainly by direct fermentation using microorganisms. Optical resolution in which N-acyl-DL-amino acids are hydrolyzed with L-aminoacylases is also a known method for producing L-amino acids. It has been utilized to industrially produce L-amino acids that are difficult to produce by fermentation. These L-aminoacylases are widely found in animals, plants, and microorganisms. They have been purified from various organisms, and their properties have been clarified. N-terminal amino acids of many proteins are considered to be N-acetylated in vivo. L-aminoacylases presumably regenerate the N-acetyl-amino acids produced by decomposition of proteins to amino acids. Among L-aminoacylases, an acylase that acts on N-acyl-L-glutamic acid is reported to be involved in arginine biosynthesis (Fruh, H., Leisinger, T.: J. Gen. Microb. 125, pp1(1981)).
In contrast, D-amino acids have not been a subject of interest for a long time because they are nonprotein amino acids. D-amino acids were known to naturally occur only in small cyclic peptides, peptidoglycan of bacterial cell walls, and peptide antibiotics. However, D-amino acids have been demonstrated to be constituents of neuro-peptides and to exist as binding forms in tooth enamel, the lens, and cerebral proteins, resulting in investigation of physiological significance and enzymatic synthesis of D-amino acids.
At present, DL-amino acids have been optically resolved by physicochemical, chemical, and enzymatic methods. The enzymatic methods are the most convenient and industrially applicable for, for example, continuously producing L-methionine from N-acetyl-DL-methionine using a bioreactor on which L-aminoacylase is immobilized. D-amino acids may also be produced using hydantoinase. The method involves a two-step enzymatic reaction. The first reaction uses D-specific hydantoinase to convert D,L-5-substituted-hydantoin, which is synthesized at low cost from aldehyde analogues, to a D-carbamyl derivative. The second reaction uses D-amino acid carbamylase. Moreover, a method is known in which D-aminoacylase hydrolyzes N-acetyl-DL-amino acids to produce D-amino acids (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980), Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang, Y. J.: J. Enzyme Microb. Technol. 14, pp384 (1992)). Thus, D-aminoacylases are important for production of D-amino acids. However, their physiologic importance and structural functions and so on remain to be resolved.
D-aminoacylase was first reported to be found in Pseudomonas sp. KT83 isolated from soil by Kameda et al. in 1952 (Kameda, Y., Toyoura, H., Kimura, Y. and Yasuda, Y.: Nature 170, pp888 (1952)). This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine, D-tyrosine, and D-alanine. Thereafter, D-aminoacylases derived from microorganisms were reported as follows:
Genus Pseudomonas (Kubo, K., Ishikura, T., and Fukagawa, Y.: J. Antibiot. 43, pp550 (1980); Kubo, K., Ishikura, T. and Fukagawa, Y.: J. Antibiot. 43, pp556 (1980); Kameda, Y., Hase, T., Kanatomo, S. and Kita, Y.: Chem. Pharm. Bull. 26, pp2698 (1978); Kubo, K., Ishikura, T. and Fukagawa, Y.: J. Antibiot. 43, pp543 (1980));
Genus Streptomyces (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42, pp107 (1978); Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980));
Genus Alcaligenes (Tsai, Y. C., Tseng, C. P., Hsiao, K. M. and Chen, L. Y.: Appl. Environ. Microbiol. 54, pp984 (1988); Yang, Y. B., Hsiao, K. M., Li, H., Yano, Y., Tsugita, A. and Tsai, Y. C.: Biosci. Biotech. Biochem. 56, pp1392 (1992); Yang, Y. B., Lin, C. S., Tseng, C. P., Wang, Y. J. and Tsai, Y. C.: Appl. Environ. Microbiol. 57, pp2767 (1991); Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H. and Wang: Microb. Technol. 14, pp384 (1992); Moriguchi, M. and Ideta, K.: Appl. Environ. Microbiol. 54, pp2767 (1988); Sakai, K., Imamura, K., Sonoda, Y., Kido, H. and Moriguchi, M.: FEBS, 289, pp44 (1991); Sakai, K., Obata, T., Ideta, K. and Moriguchi, M.: J. Ferment. Bioeng. 71, pp79 (1991); Sakai, K., Oshima, K. and Moriguchi, M.: Appl. Environ. Microbiol. 57, pp2540 (1991); Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T. and Wakayama, M.: Biosci. Biotech. Biochem., 57, pp1145 (1993); Wakayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K. and Moriguchi, M.: J. Biochem. 118, pp204 (1995)); Moriguchi, M., Sakai, K., Miyamoto, Y. and Wakayama, M.: Biosci. Biotech. Biochem., 57, pp1149 (1993));
Genus Amycolatopsis (Japanese Patent Application No. Hei 9-206288);
Genus Sebekia (Japanese Patent Application No. Hei 10-089246); and
fungus (Japanese Patent Application No. Hei 10-228636).
Tsai et al. and Moriguchi et al. also clarified the characteristics of D-aminoacylase derived from microorganisms belonging to the genera Alcaligenes and Pseudomonas and the amino acid and nucleotide sequences of the enzymes. Moriguchi et al. found, by using different inducers, three types of D-aminoacylases in microorganisms belonging to the genera Alcaligenes and Pseudomonas (Wakayama, M., Katsumo, Y., Hayashi, S., Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech. Biochem. 59, pp2115 (1995)).
Furthermore, Moriguchi et al. determined the nucleotide sequences of these D-aminoacylases derived from a microorganism belonging to the genus Alcaligenes and compared them with L-aminoacylases derived from Bacillus stereothermophilus, human, and pig. The results demonstrated that these D-aminoacylases have a low homology with L-aminoacylases (Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech. Biochem., 59, pp2115 (1995)).
As to Actinomycetes, Sugie et al. reported D-aminoacylase of a microorganism belonging to the genus Streptomyces (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42, pp107 (1978), Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980)). However, the enzyme has not yet been purified, and its characteristics have not been well clarified.
The thermal stability of any of these known D-aminoacylases above are below 50° C., and the optimal temperature is below 50° C. No D-aminoacylases with higher thermal stability is presently known. It is economically advantageous to use heat-stable D-aminoacylases, since durability of the enzyme rises with thermal stability. Moreover, application of heat-stable D-aminoacylases in the production of D-amino acid possesses economic merit as well, since it is possible to set the reaction temperature high enough to elevate the concentration of the substrate and such due to the higher solubility.